Method for producing pearl aragonite-like multilayer nanostructures using yeast

Abstract

The objective is to provide a method for producing functional nanomaterials, particularly calcium carbonate crystals having aragonite-like multilayer nanostructures, by controlling the biomineralization process. [Solution] We have demonstrated that the formation of calcium carbonate crystals can be controlled by culturing recombinant yeast or recombinant filamentous fungi, modified to express at least one protein derived from the mantle secretion of the mabe snail (Pteria penguin), in a medium containing calcium salts. By using recombinant yeast or recombinant filamentous fungi, multilayer nanostructure crystals can be produced.

Description

Technical Field

[0001] The present invention relates to biomineralization, particularly to a method for producing calcium carbonate crystals using mavetectin.

Background Art

[0002] Biomineralization refers to the phenomenon in which organisms generate minerals and inorganic substances through metabolic activities. Biominerals are produced in various organisms, from microorganisms to higher animals. Specifically, hydroxyapatite, which is a type of calcium phosphate that forms the bones and teeth of animals, and calcium carbonate, which is the main component of shells and eggshells, are biominerals.

[0003] It has been found that biomineralization is carried out through a process in which the crystal structure and shape are highly controlled by specific proteins and organic molecules, but the process is often unclear. Elucidating the process of crystal structure formation is highly anticipated because the crystals generated by biomineralization may add value to nanoscale components such as biosensors and biominerals such as pearls.

[0004] Mollusks, which are soft-bodied animals, form shells composed of calcium carbonate as described above. In the marine environment where many mollusks inhabit, calcium salts and bicarbonate ions are abundant, so calcium carbonate is widely used by organisms. Therefore, the calcium carbonate skeleton is the most abundant and common biomineral.

[0005] The mabe oyster (Pteria penguin) is a bivalve mollusk belonging to the same family as the Akoya oyster, which is used in pearl cultivation, and is utilized in the production of cultured pearls. Generally, natural pearls are formed when a layer of calcium carbonate is secreted to coat irritants and foreign matter inside the shell. Shells and pearls are mainly composed of three different anhydrous crystalline polymorphs: vaterite (hexagonal), aragonite (orthorhombic), and calcite (rhombohedral). The calcium carbonate in pearls crystallizes as aragonite, which is the main material of the pearl.

[0006] The crystallization process and its rate greatly influence the size, shape, and luster of pearls. In pearl farming, several techniques are used to optimize the crystallization of calcium carbonate. Environmental factors such as water temperature, salinity, and nutrients are used to control the crystallization process and obtain pearls that are more uniform in shape and size. However, problems have been pointed out regarding the production of cultured pearls, such as the potential for water pollution and disruption of ecosystems, and the lack of diversity in shape, luster, and color compared to natural pearls. If the crystallization of calcium carbonate can be controlled artificially, it is thought that it would have diverse applications not only in the production process of cultured pearls but also as a functional nanomaterial.

[0007] Attempts are being made to fabricate functional nanomaterials using biomineralization. Patent Document 1 discloses a method for obtaining silica nanoparticles using E. coli by utilizing diatom biomineralization.

[0008] In the biomineralization process of marine organisms, lectins have been shown to be closely involved, creating templates and scaffolds that promote crystal growth. It has also been revealed that proteins are intricately mixed within mineral structures such as the exoskeleton of corals and the shells of mollusks (Non-Patent Document 1). Patent Document 2 discloses a method of forming a three-dimensional structure (multilayer structure) by repeatedly attaching inorganic material-binding peptides as a single layer to the surface of an inorganic substrate, utilizing its biomineralization ability to form a biomineral layer, and then attaching more inorganic material-binding peptides to the surface and utilizing its biomineralization ability to form another biomineral layer. In other words, it has been shown that not only inorganic materials but also organic substances such as inorganic material-binding peptides are important for forming multilayer structures.

[0009] By analyzing the role of proteins such as lectins in crystal formation, it may be possible to form a desired crystal structure. The inventors of this invention have also analyzed proteins contained in the mantle secretion of the mabe oyster and found that it contains jacalin-related β-prism fold lectins (JRL), ART-26P-1, ART-26P-2, and ART-26P-3, which are ADP-ribosyltransferase-like 26kDa protein family proteins, as well as CA1 and CA2, which are carbonic anhydrase family proteins.

[0010] Jacalin-related lectins are multiple lectins that exhibit 35-50% homology to each other, obtained by purifying mantle secretions by affinity chromatography. They are named jacalin-related lectins because they show homology to jacalin, a lectin extracted from jackfruit seeds, and human ZG (Zymogen granule membrane protein) 16 (Non-patent documents 2, 3).

[0011] The present inventors have shown that jacarin-related lectins derived from the mantle secretion of the mabe oyster contain four types of lectins: PPL2A (PPL2αγ heterodimer), PPL2B (PPL2ββ homodimer), PPL3A / 3B / 3C (PPL3 αα / αβ / ββ dimer), and PPL4 (PPL4αβ heterodimer), and that they generate polycrystalline material in vitro (Non-Patent Literature 4). [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] Japanese Patent Publication No. 2006-197825 [Patent Document 2] International Publication No. 2006 / 126595 [Non-patent literature]

[0013] [Non-Patent Document 1] Flores, RL, et al.. BMC Evol Biol 17, 125, 2017, 10.1186 / s12862-017-0978-z. [Non-Patent Document 2] Naganuma T. et al. PLoS One, 9(11), e112326, 2014,10.1371 / journal.pone.0112326. [Non-Patent Document 3] Ogawa T. et al., Int J MolSci, 20(18), 4629, 2019,10.3390 / ijms20184629. [Non-Patent Document 4] Ogawa T. et al., Int J MolSci, 22(3),1081, 2021, 10.3390 / ijms22031081. [Overview of the project] [Problems that the invention aims to solve]

[0014] If the process of biomineralization can be elucidated and the resulting crystal structure can be controlled, it will become an extremely useful technology in the manufacture of functional nanomaterials and the like. According to Patent Document 1, low-cost silica particles can be formed using E. coli, but they do not have a multilayer structure and are not a technology for controlling the crystal structure. The method described in Patent Document 2 can form a multilayer structure, but it is necessary to repeat the process for each layer, making the operation complicated.

[0015] Although the inventors were able to form crystals in the mabe oyster as described above, they were unable to form a multilayer structure like that of actual pearls. The object of this invention is to provide a method for controlling the formation of crystals using mabe oyster lectin and forming a biomineral having a multilayer structure. This method makes it possible to provide functional nanomaterials at low cost and with simple operation. [Means for solving the problem]

[0016] This invention relates to a method for producing the following multilayer nanomaterials. (1) A method for controlling calcium carbonate crystals, characterized by culturing recombinant yeast or recombinant filamentous fungi, which have been modified to express at least one protein derived from the mantle secretion of the mabe oyster (Pteria penguin), in a medium containing calcium salts. Previously, researchers expressed mantle secretion-derived proteins in E. coli and analyzed their crystal structures, but were unable to produce crystals with multilayer nanostructures. However, when mantle secretion-derived proteins were expressed in yeast, multilayer crystal structures were produced on the surface of yeast cells. It is thought that similar multilayer crystals can be produced in fungi that possess a chitin synthesis system, such as filamentous fungi.

[0017] (2) A pearly multilayer nanostructure calcium carbonate crystal characterized by containing a protein derived from the mantle secretion of the mabe oyster, which is expressed by the introduced gene. Calcium carbonate crystals with a pearl-like multilayer nanostructure produced by yeast or filamentous fungi contain a protein derived from the mantle secretion fluid expressed by the introduced gene. Although it is known that pearls also contain other proteins such as MSI60 and perlin (N16), artificially created crystals contain only the introduced protein, so they can be distinguished.

Brief Description of Drawings

[0018] [Figure 1A] A diagram schematically showing the structure of the introduced plasmid. [Figure 1B] A diagram schematically showing a method for expressing mabe lectin in yeast and producing multilayer nanostructure crystals. [Figure 2] A diagram showing the results of analyzing protein expression by Western blotting. The left side of the figure shows the analysis results of PPL2A expression, the upper part shows PPL2α, the lower part shows PPL2γ, the right side shows the analysis results of PPL4 expression, the upper part shows PPL4α, and the lower part shows the expression of PPL4β. [Figure 3A] An optical micrograph image showing an example of a protocrystal, which is the crystal form at the initial stage. [Figure 3B] An optical micrograph image showing an example of the observed square crystal. [Figure 3C] An optical micrograph image showing an example of a large composite structure composed of a large number of small crystals. [Figure 3D] An optical micrograph image showing an example of a crystal with a fan-shaped structure. [Figure 3E] A diagram schematically showing the crystal growth process. [Figure 4] A scanning electron micrograph showing the spatial relationship between the forming crystal and yeast cells. [Figure 5] A diagram showing the results of elemental composition analysis using energy-dispersive X-ray analysis (EDX). [Figure 6] A diagram showing the results analyzed by X-ray diffraction method. [Figure 7] A diagram showing the number of formed crystals cultured under different pH culture conditions. [Figure 8]A schematic diagram illustrating the lectin-mediated crystal formation mechanism in yeast. [Modes for carrying out the invention]

[0019] Figure 1B schematically shows a method for expressing mabegay lectin in yeast and fabricating multilayer nanostructures. Here, mabegay lectins PPL2A and PPL4 are expressed in yeast to form multilayer calcium carbonate crystals, but similar multilayer structures can be formed by expressing other mabegay lectins, specifically PPL2B, PPL3A / 3B / 3C, in yeast.

[0020] Furthermore, conventional methods could form crystals, but could not form multilayer crystals. Multilayer crystals were first successfully formed in yeast, which is thought to be because yeast synthesizes chitin, and chitin is present in its cell wall. Therefore, it is possible to similarly form multilayer crystals using fungi that possess a chitin synthesis system, such as filamentous fungi.

[0021] [Plasmid Production and Preparation] The manufacturing method will be explained below, step by step, with accompanying data. Here, the plasmid was prepared using E. coli DH5α, and the protein was expressed in Saccharomyces cerevisiae to form crystals. After examining various strains of Saccharomyces cerevisiae, we decided to use the RSY620 strain (provided by the Randy Schekman Laboratory at the University of California, Berkeley), which showed high expression. The cultivation of E. coli, plasmid preparation, and yeast culture can be carried out using commonly used techniques.

[0022] Since PPL2A is a heterodimer of PPL2α (SEQ ID NO: 1) and PPL2γ (SEQ ID NO: 2), and PPL4 is a heterodimer of PPL4α (SEQ ID NO: 3) and PPL4β (SEQ ID NO: 4) (Non-Patent Documents 2 and 3), constructs were created using the bidirectional expression plasmid pBEVY-U or pBEVY-L (addgene) to express both proteins in yeast (Figure 1A).

[0023] To enable detection of protein expression in subsequent steps, HA tags and Myc tags were added to the PPL4α and PPL4β proteins, respectively, by PCR using the following primers. Furthermore, the two subunits of the PPL2A protein, PPL2Aα and PPL2Aβ, were cloned without adding detection tags due to their high affinity for the monoclonal antibody.

[0024] PPL4α-HA-fwd:5'-TAGTTTCGACGGATCCAAAAAATGTTCATAATATTCAAGTGTACCAGCAGAA-3'(Sequence ID 5) PPL4α-HA-rev:5'-CAAAGCTTGCATGCCTGCAGTTACGCATAGTCAGGAACATCGTATGGGTACCACAATGGCCAGTAAAACTGTATTCT-3' (SEQ ID NO: 6) PPL4β-Myc-fwd:5'-ACTCCCCGGGTACCGAGCTCAAAAAATGGGTTTCTATGTGTACATTGTTCTCCT-3'(Sequence ID 7) PPL4α-HA-rev:5'-GAAGTGTCGAATTCGAGCTCTTACAGATCTTCTTCAGAAATAAGTTTTTGTTCCCACGATGGCCAGTAGAACTG-3' (SEQ ID NO: 8)

[0025] The prepared pBEVY vector and the insert DNA prepared by PCR were cloned using an in-fusion cloning system. The insertion of the desired DNA fragment was confirmed by sequencing, and these were then used in subsequent experiments. Plasmids expressing each subunit individually were prepared in the same manner. The plasmids were prepared using E. coli.

[0026] [Protein preparation] Plasmid transformation of budding yeast was performed using the lithium acetate method. The single colonies obtained by transformation were inoculated into 10 mL of SD liquid medium (0.67% yeast culture nitrogen base (amino acid-free), 0.062% yeast dropout medium additive (-LWU), 0.002% tryptophan, 1% glucose) and incubated overnight at 30°C and 200 rpm until the OD600 reached 4.0. The culture solution was transferred to a 1 mL Eppendorf tube and centrifuged at 13,000 rpm for 1 minute to collect the culture solution. After removing the supernatant, 40 μL of B88 buffer (20 mM Hepes pH 6.8, 250 mM Sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT, 2% protease inhibitor mix, 2 μg / mL Pepstatin A) was dispensed, glass beads were added until they reached the solution surface, and the mixture was vortexed at 4°C for 1 minute, followed by 1 minute on ice. This process was repeated 10 times to extract the protein.

[0027] The bottom of an Eppendorf tube was punctured with a 25G x 1 needle, and the tube was placed on top of a new Eppendorf tube and centrifuged at 3,000 rpm for 2 minutes at 4°C. Considering the difficulty of disrupting the yeast cell wall, a bioruptor was used to disrupt the cell wall for 30 seconds, followed by 30 seconds of placement on ice, repeated for 20 minutes. After completion, the tube was centrifuged at 7,000 rpm for 2 minutes to separate the supernatant from the precipitate. After adding 40 μL of B88 buffer to the precipitate, 10 μL of 6 x SDS sample buffer was added to 10 μL of the resulting sample (culture medium / supernatant / precipitate), and the mixture was heated at 95°C for 5 minutes to prepare an SDS-PAGE sample.

[0028] [Analysis using Western blotting] SDS-PAGE was performed using a 12.5% ​​acrylamide gel according to a standard method, followed by analysis by Western blotting. Proteins were transferred using a wet transfer method on a 0.45 μm PVDF membrane (Immobilon-P, Millipore). After transfer, the PVDF membrane was blocked with TBS-T (Tris-buffered saline containing 0.1% Tween 20) with 5% skim milk for 45 minutes at room temperature. Subsequently, it was washed with TBS-T and reacted overnight with a primary antibody diluted 1:5000 at 4°C. The membrane was washed with TBS-T by shaking three times for 5 minutes, reacted with a secondary antibody diluted 1:5000, reacted at room temperature for 45 minutes, washed with TBS-T three times for 5 minutes, and detected by chemiluminescence. A luminescent image analyzer (LAS-4000, FUJIFILM) was used as the detector. The detected bands were quantified using a CS Analyzer 3.0.

[0029] Furthermore, PPL2Aα and PPL2Aγ proteins were detected using highly specific mouse polyclonal antibodies as primary antibodies. PPL4α and PPL4β, tagged with HA or Myc, were detected using α-HA (12CA5, Sigma-Aldrich, 1:2000 dilution) and α-Myc (Novex, 1:5000 dilution) as primary antibodies.

[0030] For secondary antibodies, when mouse polyclonal antibodies were used as primary antibodies, rabbit anti-mouse IgG antibody (HRP-labeled) (Cell Signaling Inc.) was used. For anti-HA antibodies and anti-Myc antibodies, peroxidase-labeled goat anti-rabbit IgG(H+L) polyclonal antibody (affinity-purified) (Funakoshi Inc.) was used diluted to 1:5000.

[0031] In Figure 2, M represents the medium, S represents the supernatant, and P represents the precipitate. Furthermore, "Vector" shows the vector alone, while "2Aα," "2Aγ," "4α-HA," and "4β-Myc" show the results of expressing PPL2Aα, PPL2Aγ, PPL4α, and PPL4β individually, respectively. "2Aα+2Aγ" shows the result of simultaneously expressing the two α / γ subunits of PPL2A, and "4α-HA+4β-Myc" shows the result of simultaneously expressing the two α / β subunits of PPL4.

[0032] Protein bands were observed at the expected molecular weights of 18.1kD (PPL2Aα), 18.4kD (PPL2Aγ), 21.1kD (PPL4α), and 20.0kD (PPL4β).

[0033] [Observation results using an optical microscope] The biomineralization process in the transformed yeast strain RSY620 was analyzed as follows: A single colony of the transformant was inoculated into 5 mL of SD liquid medium and cultured at 30°C and 200 rpm until the OD600 reached 0.6. 0.64 g / L Ca 2+ Transgenic plants were cultured in 50 mL of B4 medium (0.4% yeast extract, 0.067% calcium acetate monohydrate) containing 1 × 10⁻¹⁶ of the transformed plants. 5 The solution was adjusted to CFU / ml. After incubation at 30°C and 150 rpm for 14 days, crystal formation was analyzed.

[0034] The obtained crystals were photographed using an optical microscope (Nikon Eclipse Ni-U). The samples were prepared as follows: After removing the supernatant, the precipitated sample was stirred using a vortex mixer to disperse any remaining liquid and homogenize the sample. Then, 1 μL of the sample was pipettered and diluted 10-fold with distilled water or buffer solution to reduce the sample concentration to a level that facilitated the identification of individual crystals and their morphologies. Using a 20x objective lens, images were taken from nine different positions on the slide, taking care to avoid being affected by variations between samples, and observations were performed accordingly.

[0035] Various crystal forms were observed, but numerous protocrystals with rough edges were seen within the field of view (Figure 3A). Protocrystals are an early stage of crystal formation, with irregular edges, suggesting that the crystals are in the process of growth or aggregation. In addition to these protocrystals, a small number of square crystals were also observed (Figure 3B). These square crystals are thought to be a specific polymorph of calcium carbonate. Under the culture conditions tested, it became clear that different crystalline structures coexisted in the sample. Furthermore, several complex structures consisting of numerous small crystals were present (Figure 3C). These aggregates are thought to have been formed by the aggregation of small crystals, probably due to surface interactions or the action of proteins and polysaccharides as binders. It is a well-known phenomenon in microbial crystallization processes that microbial surfaces function as nucleation sites for crystal growth, forming larger and more complex polycrystalline aggregates. Furthermore, numerous crystals with fan-shaped and clover-shaped structures were observed (Figure 3D). The characteristic fan shape is thought to be an intermediate form in the formation of vaterite, one of the polymorphs of calcium carbonate. These intermediate forms are known to arise in biological and environmental crystal formation processes, preceding the transition to the more common and stable spherical form of vaterite. Figure 3E shows a schematic diagram illustrating the crystal growth process of vaterite (upper panel of Figure 3E) and observed crystals thought to be at each stage of crystal growth (lower panel of Figure 3E). The observed crystal structures actually show images that are thought to represent the crystal formation process.

[0036] [Observation using a scanning electron microscope] Because different polymorphs can exhibit similar forms, the composition and crystal structure cannot be determined solely by the shape of the crystal. Accurate determination of the composition and crystal type requires analysis using methods such as scanning electron microscopy (SEM) or X-ray diffraction (XRD). The results of the SEM analysis are shown first.

[0037] In addition to analyzing the crystal structure, we observed the morphology of cultured yeast precipitates using a scanning electron microscope to investigate the spatial relationship between crystals and yeast cells during crystal formation. A HITACHI SU8000 scanning electron microscope was used, and observations were performed at an acceleration voltage of 3.0 kV.

[0038] After culturing, yeast and crystals were collected, and scanning electron microscope samples were prepared using conventional methods. In samples prepared immediately after collection of cultured yeast and crystals, the yeast cells retained their original morphology, maintaining complete cellular form. Therefore, the crystal formation location and its relationship with the yeast cells could be accurately observed, and insights into the mechanism of crystal formation were obtained.

[0039] As shown in Figure 4, numerous small crystals aggregate between yeast cells, forming irregular structures and scattered throughout the cells. Different crystal structures were observed, thought to depend on the expressed mabegay lectin and the stage of crystal growth. These included square crystals induced by the PPL2Aα protein, yeast cells surrounded by crystals induced by the PPL2Aγ protein, large crystals induced by the PPL2A protein with many yeast cells attached to the surroundings (top row), rhomboid and polygonal crystals induced by the PPL4α-HA protein, square crystals induced by the PPL4β-Myc protein (middle row), and square crystals (bottom left) and layered structures (bottom center, right) seen with the PPL4-HA-Myc protein. The spatial distribution of mabegay lectin and the localization of protein expression suggest that crystal formation is highly likely to depend on the action of lectin proteins localized on the cell membrane by signal peptides.

[0040] The observed crystals were most likely formed directly on the surface of yeast cells. Chitin, a polysaccharide found in the exoskeletons of arthropods and insects, and in fungi, is known to provide a substrate for nucleation and growth of calcium carbonate crystals during the precipitation of calcium carbonate induced by microorganisms. The observation of a multilayered crystal structure in yeast suggests that chitin, one of the components of the yeast cell wall, plays an important role. Since filamentous fungi also have chitin in their cell walls, it is highly likely that multilayered crystals can be similarly produced in filamentous fungi. As filamentous fungi, Aspergillus nidulans, which is easy to genetically modify, and Aspergillus oryzae, Aspergillus sojae, and Aspergillus luchuuensis, which do not contain aflatoxins and are used in the brewing industry, can be used.

[0041] Although the data is not shown here, in samples that were air-dried for a long period on an SEM sample stage, the morphology of the yeast cells was destroyed by the prolonged drying, making it impossible to accurately identify the relationship between the crystal formation site and the yeast cells. However, the prolonged drying made the distribution of the crystals themselves clearer, allowing for clearer observation of the crystal morphology. A small number of rhomboid crystals and a large number of spherical crystals were observed in the samples.

[0042] [Energy-dispersive X-ray spectroscopy] Scanning electron microscopy observations indicate that yeast cells play a central role in promoting nucleation and growth of crystal structures, suggesting that chitin and lectin proteins are involved in mediating crystal formation on the cell surface. To fully characterize the crystals and confirm the mechanism of crystal precipitation by yeast, structural and compositional analysis was performed by X-ray diffraction.

[0043] Elemental composition analysis was performed using energy-dispersive X-ray spectroscopy (EDX). For EDX, a HITACHI SU8000 scanning electron microscope (HITACHI Corporation) equipped with an energy-dispersive X-ray analyzer (AMETEK Corporation) was used, specifically the SUTW (super ultra-thin polymer window) configuration. For EDX analysis, a portion of the crystal was randomly selected from SEM capture images, coated with a 10 nm thick carbon layer, and air-dried polished samples were used to record spectral maps (256 × 200 pixels) at an acceleration voltage of 15 kV and a microscope magnification of 5960x. From these spectral maps, the cumulative spectrum of the crystalline region was calculated, and a background subtraction map was created using EDAX Genesis software (Figure 5).

[0044] Energy-dispersive X-ray spectroscopy analysis of a portion of the crystals in the samples revealed characteristic peaks corresponding to various elements such as calcium (Ca), carbon (C), and oxygen (O) atoms in all samples. The most prominent peak in the spectrum corresponds to the Kα X-ray emission of calcium, indicating the presence of calcium within the crystal structure. Furthermore, each sample differed in composition from that of seashells and possessed a unique peak pattern.

[0045] In EDX analysis of calcium carbonate crystals, the peak corresponding to calcium was more pronounced than the peaks corresponding to carbon and oxygen. This is thought to be because calcium has an atomic number of 20, which is larger than that of carbon (atomic number 6) and oxygen (atomic number 8), resulting in stronger X-ray emission and thus a stronger calcium peak. EDX analysis has also been performed in other biomineralization studies, and these results show a similar profile.

[0046] [Analysis by X-ray diffraction] The crystalline phase of the product was identified using X-ray diffraction (XRD). XRD measurements were performed using a Rigaku Rint2200 with Cu Kα radiation (wavelength 1.54187 Å) and an output of 1.6 kW (40 kV, 40 mA) as the incident X-ray. The cultured sample was poured into a 50 ml tube, centrifuged at 420 rpm for 20 minutes, the supernatant was discarded, and the precipitate was placed in a glass XRD sample holder (22 mm × 22 mm), where the surface was gently pressed to smooth it. After sealing the sample surface with polyimide tape, it was analyzed by XRD. Scanning was performed in a 2θ range from 15° to 60°. The obtained XRD patterns were then analyzed using Igor Pro 8 software and compared with a PDF (powder diffraction file) database for reference.

[0047] As shown in Figure 6, vaterite was the main crystalline phase of the formed calcium carbonate crystals. The XRD pattern shows several sharp diffraction peaks characteristic of the hexagonal structure unique to vaterite. The most prominent peaks were observed around 2θ angles of 23.4°, 28.9°, and 31.4°, corresponding to the (101), (110), and (111) interplanar spacings of vaterite, respectively. These peaks were observed relatively clearly in samples where crystallization had progressed. In addition, a broad peak was observed in the 2θ range of 15° to 22°, which does not correspond to the crystalline structure of vaterite. This is thought to be due to the presence of polyimide tape and yeast used in sample preparation.

[0048] XRD patterns revealed that the peak intensity of the PPL2A protein, particularly PPL2Aα, was strong, while the peak intensity of the PPL4 protein was remarkably low. This observation suggests that the PPL2A protein more effectively promotes the crystallization of calcium carbonate, resulting in higher crystallinity and a more orderly crystal structure.

[0049] [Quantification of crystals] To quantify the crystals, images of the crystal samples were taken using an optical microscope, and then the images were processed using ImageJ software. Specifically, ImageJ's grayscale analysis and differential functions were used to enhance the contrast between the crystals and the background, allowing for more accurate identification of crystals within the images. All processed images were input into the WEKA program, which incorporates a counting plugin designed for automated analysis, to count the crystals and efficiently quantify the number of crystals present in the sample while ensuring accuracy.

[0050] Samples were prepared by culturing yeast cells under different pH conditions, pH 6 and pH 7.8. The number of crystals detected in the sample obtained at pH 6 was significantly higher than in the sample obtained at pH 7.8 (Figure 7). This difference is thought to be due to the inhibitory effect of alkaline conditions on yeast growth. Yeast cells, particularly Saccharomyces cerevisiae, are known to experience growth inhibition in alkaline environments. High pH is said to impair metabolic processes, including protein synthesis and cell division, leading to decreased cell proliferation. Furthermore, crystal nucleation sites are considered important factors in calcium carbonate precipitation. Because the cell surface of yeast cells is negatively charged, Ca ions accumulate on the cell surface and act as calcium carbonate nucleation sites. Therefore, under pH 6 culture conditions, yeast cells proliferate more effectively, resulting in a larger number of cells available as nucleation sites for crystal formation. In contrast, at pH 7.8, yeast growth is restricted, the number of proliferating cells decreases, and the number of nucleation sites available for crystal growth decreases.

[0051] Figure 8 schematically illustrates the mechanism of lectin-induced crystal formation in yeast. Aerobic respiration and oxidation of organic acids in yeast (Saccharomyces cerevisiae) lead to an increase in pH and dissolved inorganic carbon (DIC). The figure shows that calcium carbonate is deposited via a matrix protein derived from mabegai lectin encoded by the introduced plasmid, forming polymorphic crystals.

[0052] As demonstrated above, we were able to form multilayer crystals by expressing mabegay lectin in yeast. This demonstrates that functional nanomaterials can be provided at low cost and with simple operations by using a method for fabricating multilayer nanostructures.

Claims

1. A method for controlling calcium carbonate crystals, characterized by culturing recombinant yeast or recombinant filamentous fungi, modified to express at least one protein derived from the mantle secretion of the mabe oyster (Pteria penguin), in a medium containing calcium salts.

2. The method according to claim 1, characterized in that the protein derived from the mantle secretion of the mabe oyster is one of the jacalin-related lectins PPL2A, PPL2B, PPL3, and PPL4.

3. The method according to claim 2, characterized in that the jacarin-related lectin family is PPL2A and / or PPL4.

4. A method for producing an aragonite-like multilayer nanostructure using the method described in any one of claims 1 to 3.

5. The manufacturing method according to claim 4, characterized in that PPL2A and / or PPL4 are distributed at the calcium carbonate polycrystalline interface to form polycrystals.

6. A pearly, multilayered nanostructured calcium carbonate crystal characterized by containing a protein derived from the mantle secretion fluid of the mabe oyster, which is expressed by the introduced gene.

7. The protein derived from the mantle secretion of the aforementioned Mabe snail, The calcium carbonate crystal according to claim 6, characterized in that it is one or more of the jacalin-related lectins PPL2A, PPL2B, PPL3, and PPL4.

Images